Overview
Our team revolves around GABA, a naturally occurring non-protein amino
acid, which has become a
major research hotspot in food science as a neurotransmitter with functions such as lowering
blood pressure, relieving anxiety and stabilizing mood. This project aims to improve GABA
content by knocking out CaM gene with overexpression of GAD gene in longan using CRISPR-Cas9
gene editing technology. Meanwhile, we also performed blue light treatment and high temperature
treatment on longan healing tissues based on the characteristics that GABA can respond to
adversity stress, all of which significantly increased GABA content. After obtaining the high
GABA longan cell line, we used this cell line for fermenter production, which is expected to
achieve factory production in the future, and extract GABA from it to better serve the
pharmaceutical industry.
Background
With the accelerated pace of people's lives, the development of electronic
science and
technology and the increase of work pressure, insomnia has become a worldwide public health
problem and a major factor inducing various mental health problems [1], and the 2020 Scientific
Sleep and Mental Health Research Report shows that sleep quality is directly linked to mental
health, and the chance of depression in insomnia patients is as high as 50%.
Image source：Visual Capitalist
More than 300 million people worldwide are expected to suffer from depression, with
a global
average
prevalence of around 4.4%. In addition, hypertension is a risk factor for heart disease and
stroke, and according to the Global Hypertension Epidemic Analysis Study, the number of adults
aged 30-79 with hypertension will increase from 650 million to 1.28 billion between 1990 and
2020. Thus, these three factors are important threats to human health.
GABA is an important
active substance in the human nervous system with potential health benefits such as
antidepressant, sedative, antihypertensive, anticancer, and antidiabetic [2], however, the
amount of GABA in the human body is limited, and intake from food is an important way to
supplement GABA, while the amount of GABA in plant tissues is low, usually between 0.3 and 32.5
μmol-g-1, and does not meet the physiological requirements of the human body [3].
Foundation
Depression
Depression is now one of the most common psychological diseases, with continuous and
long-term
low mood as the main clinical characteristics, is the most important type of modern
psychological diseases.According to the Depression and Other Common Mental Disorders (Depression
and Other Common Mental Disorders) report released by the World Health Organization (WHO) in
2017, more than 300 million people worldwide are expected to suffer from depression, with a
global average incidence of around 4.4%.In terms of region, age and gender, the report also has
the following findings:
1 The incidence was higher in women than in men.The average incidence
rate in women was 5.1%, higher than 3.6% in men;
2 The incidence rate increases with age.The
prevalence of depression exceeded 5.5% in men aged 55 – 74 years and over 5.5% in women aged 55
– 74 years was over 7.5%.Women aged 60-64 years are a high-risk group, with an incidence rate of
nearly 8%;
3 The incidence is higher in low-income countries than in other
countries.[1] Data
Median mental health expenditure per capita, by World Bank income group showed significant
differences in per capita mental health spending between high-income countries and low-income
countries, close to $59 and below $2, respectively.The lack of per capita economic distribution
of mental health in LMICs governments has prevented the majority of patients from accessing
appropriate treatment resources.
Longan
Longan is an important evergreen economic fruit tree in China. Longan fruit pulp is
rich in
nutrients, such as protein and vitamin C; it also has biological activities such as antioxidant.
Inside the fruit, the content of GABA in longan is relatively high.
Fujian province is one of
the main production areas of longan and one of the main fruits in Fujian.
With the continuous
development of science and technology, the varieties of longan have been improved and its
planting area is growing rapidly. The study of GABA content of longan is of great importance to
facilitate the promotion of longan cultivation and to serve people such as insomnia as well as
depression patients.
GABA, which is present in very small amounts in plant tissues Ingestion from
food is the main way to supplement GABA, and the content of GABA in plant tissues is relatively
low, usually between 0.3 and 32.5 μmol-g-1, which cannot meet the physiological requirements of
the human body [4].In longan, the content of GABA was significantly higher than 32.5 μmol-g-1,
and different longan varieties,There are also large differences in GABA content, which may be
closely related to their environmental and other conditions [5].GABA is involved in various
metabolic pathways and processes as a signaling molecule, and has a regulatory role in insomnia,
depression and hypertension.
Our research team, Zhongxiong Lai, has deciphered the genome of
longan and published it online in the journal Giga Science published by Oxford University Press.
This is the first international publication of the whole genome database of longan, and the
first whole genome database of Sapotaceae. It is also the first whole-genome database of
Sapotaceae, which marks our research in related fields at the international leading level [6].
We also established a rapid transgenic method for longan, which has the advantages
of easy
operation, low cost, short cycle time, high transformation efficiency, high survival rate, and
annual production. Based on the research basis in the laboratory, this experiment aimed to
investigate the organelles that can produce GABA efficiently by constructing overexpression
vectors, gene cloning, and experimental methods such as adversity stress.
GABA pathway
In the presence of GAD, glutamate undergoes decarboxylation to form GABA, which is
then
catalyzed by GABA transaminase (GABA- T) to form succinic semialdehyde. Finally, succinic
semialdehyde dehydrogenase (SSADH) catalyzes the oxidative dehydrogenation of succinic
semialdehyde to form succinic acid, which eventually enters the tricarboxylic acid cycle. In
this process, GAD is the rate-limiting enzyme for the synthesis of GABA. GAD in plants contains
a calmodulin (CaM) binding region, and GAD activity is co-regulated by Ca2+ and H+
concentrations. At the same time, GAD activity is also influenced by the concentration of the
GAD coenzyme pyridoxal phosphate (PLP) as well as the substrate glutamate. This dual regulatory
mechanism links the cellular accumulation of GABA to environmental stress , where cold stress,
heat stress, osmotic stress, and mechanical injury can increase GAD activity and enrich GABA.
Meanwhile, the use of gene editing to add or delete genes related to this pathway is another
approach to enrich GABA.
In summary, based on the characteristics of GABA metabolic pathway and response to
adversity
stress, our team obtained a high GABA cell line by constructing overexpression vector, gene
editing technology with adversity stress treatment and other experimental methods. We used this
cell line for fermenter production and extracted GABA from it to solve people's insomnia,
depression, low mood and other troubles.
References
[1]LU Xiao-fengSUN LingZHANG Hui-zhen et,al.Investigation Analysis of Sleep Disturbances
Epidemiology[J].Gansu Journal of Traditional Chinese
Medicine,2011,24(2):67-69.DOI:10.3969/j.issn.1004-6852.2011.02.035
[2]ABDELAZEZO A, ABDELMOTAAL H, EVIVIE S E, et al.
Screening potential probiotic characteristics of lactobacillus brevis strains in vitro
and intervention effect on type I diabetes in vivo [J].
Bio Med Research International, 2018, 1-20.
[3]MIAO J W. Summary ofγ-aminobutyric acid[J]. The Food Industry, 2021,42(4):363-366.
[4]Sun Xing, Wang Wenwen, Wei Chunjie, Lu Shuang, Zheng Zhiren, Zhang Hui, Xu Chenxi, Wang
Chong. Research progress of using gene editing technology to improve GABA content in tomato[J].
Journal of Shanghai Normal University (Natural Science Edition),2022,51(01):87-93.
[5]ZHENG Shaoquan, JIANG Fan, HUANG Aiping, GAO Huiying, DENG Chaojun, CHEN Xiumei, ZHENG
Jingui. Analysis of γ-aminobutyric acid content in longan fruit of different genotypes[J].
Fujian Fruit Tree,2008(03):23-27.
[6]Scientists from Fujian Agriculture and Forestry University decipher the longan genome[J].
World Tropical Agricultural Information,2017(Z2):14-15. 4) Chen Yu-Kun, Cheng Chun-Zhen, Zhang
Z-Hao, Lai Zhong-Xiong. Establishment of a rapid genetic transformation method for longan[J].
Journal of Applied and Environmental
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